Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on the pulmonary tissue of patients with cystic fibrosis (CF), resulting in chronic infections. Longitudinal genomics studies of P. aeruginosa isolates from individual CF patients indicate that the bacterial strains are often clonal over time. This suggest that subpopulations of the original founder strains survive the antibiotic treatments, then repopulate the biofilms when treatments are alleviated. The surviving bacteria are termed persister cells. Persister cells may be metabolically dormant, and arise in part because biofilms contain microenvironments with low oxygen or nutrient availability. In order to eliminate chronic infections, it will be necessary to either prevent the formation of persister cells or prevent thei resuscitation from dormancy. Here, we will focus on the latter approach, by characterizing the role of factors that allow prolonged survival of bacteria under dormant conditions. Using laser capture microdissection (LCM) and transcriptomics, we identified mRNA transcripts that are abundant in the dormant, antibiotic-tolerant subpopulations of P. aeruginosa biofilms. These transcripts code for ribosome hibernation factors, HPF and RMF, which have been shown in Escherichia coli to convert active ribosomes to resting 100S dimers. Since many antibiotics target translation, bacteria with inactivated ribosomes would be tolerant to these antibiotics. In addition, our preliminary studies demonstrate that the absence of these factors in P. aeruginosa results in loss of cell viability during non- growth conditions. Loss of HPF results in almost complete degradation of the 23S rRNA of the 50S ribosomal subunit. Since ready-made ribosomes are necessary for cell recovery from dormancy, these factors likely evolved to protect ribosomes from complete degradation during starvation conditions. Therefore, the goals of this research are to characterize further the expression and activity of these ribosome hibernation factors and determine their role in recovery of P. aeruginosa biofilm cells from dormancy. In this research we will: (i) characterize the molecular activities of RMF and HPF, and determine their role in ribosome abundances and survival of the P. aeruginosa dormant biofilm subpopulations, (ii) characterize the transcriptional regulation of rmf and hpf, to identify factors required for teir high expression in biofilms, and (iii) determine the role of mRNA folding and other post-transcriptional regulatory processes in the expression of these factors in biofilms. Ultimately, we will identify molecular mechanisms that allow recovery of biofilm cells from dormancy. These mechanisms may then be used as targets to kill the dormant biofilm bacteria that cause chronic P. aeruginosa pulmonary infections.